Telomeres are specialized regions of non-coding repeated DNA sequences bound by proteins which form condensed, heterochromatic structures at the ends of chromosomes. During cell division, the process of DNA replication at the ends of chromosomes is imperfect, and a special enzyme known as telomerase is required to replicate telomeric DNA sequences. With each round of replication, telomeres become progressively shorter, and after approximately 50 cell cycles, the shortened telomeres cease to function, leading to cell death; thus, shortening of telomeres is associated with aging. Another indication that telomere function is associated with cellular longevity comes from the observation that 90% of immortalized tumor cells exhibit enhanced telomerase activity (Hwang, Mech. Ageing Dev. 123(12):1681-94, 2002; Perry & Jenkins, 1999). In addition to protecting chromosomal ends from “erosion” during replication, telomeres also protect chromosomal ends from degradation by nucleases. Interfering with the process of telomere replication by telomerase and thereby diminishing the protective activity of telomeres may be an effective means of speeding up cellular senescence.
The DNA found within telomeres contains tandem repeats of simple motifs such as (5′-TTAGGG)n in Homo sapiens and (5′-TTGGGG)n in the ciliate, Tetrahymena (Herbert et al., 1999; Hemann & Greider, 1999), and these DNA sequences are known to adopt an unusual, highly stable structure formed by Hoogsteen base-pairing between guanine residues. These four-stranded guanine-rich DNA molecular structures are known as G-quadruplexes, DNA tetraplexes or G-quartets. G-quadruplexes are believed to be associated with switch recombination during the differentiation of B lymphocytes, as well as being involved in gene regulation and disease states such as cancer and Werner's syndrome (Simonsson, Biol. Chem., 382, 621-628; 2001). G-quadruplexes are stabilized by physiological concentrations of potassium ions, and have been shown to directly inhibit the activity of telomerase, implicated in tumorigenesis (Davies & Siu, 2000; Elmore & Holt, 2000). Thus, compounds which stabilize G-quadruplexes and interfere with telomerase activity may serve useful as antitumor agents by causing telomere instability and combating the uncontrolled cellular proliferation observed in cancer (Riou, PNAS, 99(5):2672-2677; 2002).
While inhibition of telomerase activity can be achieved by several approaches, including altering the enzyme's RNA template or interactions with its reverse transcriptase active site, numerous studies have focused on stabilizing the inhibitory G-quadruplex structure formed by the telomeres. Molecules studied for their ability to stabilize G-quadruplex structures include the classic DNA intercalators ethidium and amido-anthraquinones. These molecules appear to drive the single-strand telomere-quadruplex equilibrium in favor of the folded quadruplex complex. In contrast, perylene derivatives and tetra-(N-methyl-pyridyl)-porphyrin appear to stack on the exterior of the quadruplex. In addition, it has been reported that a series of porphyrin derivatives demonstrate selectivity between three possible G-quadruplex types. Thus, identifying and characterizing the stability of the ligand-G-quadruplex complex remains a challenge. In addition, all so-called G-quadruplex promoters thus far reported demonstrate some affinity for duplex-DNA and tend to exhibit cytotoxicity in addition to the desirable telomerase inhibitory properties.
Given the link between G-quadruplex stabilization and inhibition of telomerase, and their presumed utility as a means of triggering senescence, it is clear that molecules that G-quadruplex stabilizers are of great potential benefit as therapeutics. To date, however, the search for such molecules has been hampered by the lack of a facile assay of G-quadruplex binding. For example, previous assays for monitoring binding of molecules to quadruplex-DNA have involved titration-NMR methodologies (Fedoroff et al., 1998; Hurley et al., 2000), relatively insensitive UV spectroscopy (Mergny et al., 1998), and time-consuming DNA-polymerase stop-assays (Han et al., 1999). While the NMR approach provides detailed structural information, the large and expensive equipment required does not lend itself to rapid screening of many potential quadruplex-binding molecules. Alternatively, an elegant time-resolved fluorescence assay using Eu3+ long-lived fluorophores bound to telomeric fragments (Bare et al., 1998), targets telomerase activity rather than quadruplex-DNA binding, and involves time-consuming PCR amplification.
Consequently, there remains a long felt need for a rapid and facile micro-method for assaying the binding of molecules to G-quadruplex DNA, and methods which facilitate the screening a library of molecules for their ability to bind and stabilize G-quadruplex structures, toward the discovery of potential therapeutics. It is clear that such a rapid and facile micro-method for assaying the binding of molecules to G-quadruplex DNA, and methods for screening molecules for their ability to stabilize the G-quadruples structure would be of great benefit in the discovery of potential therapeutics. This invention is directed to these, as well as other, important ends.